Extensive sharing of chloroplast haplotypes among East Asian Cerris oaks: The imprints of shared ancestral polymorphism and introgression

Abstract Shared ancestral polymorphism and introgression are two main causes of chloroplast DNA (cpDNA) haplotype sharing among closely related angiosperms. In this study, we explored the roles of these two processes in shaping the phylogeographic patterns of East Asian Cerris oaks by examining the geographic distributions of randomly and locally distributed shared haplotypes, which coincide with the expectations of shared ancestry and introgression, respectively. We sequenced 1340 bp of non‐coding cpDNA from Quercus acutissima (n = 418) and Q. chenii (n = 183) and compiled previously published sequence data of Q. variabilis (n = 439). The phylogenetic relationships among haplotypes were examined using a median‐joining network. The geographic patterns of interspecifically shared haplotypes were assessed to test whether nearby populations have a higher degree of interspecific cpDNA sharing than distant ones. We identified a total of 27 haplotypes that were grouped into three non‐species‐specific lineages with overlapping distributions. Ancestral haplotypes were extensively shared and randomly distributed across populations of the three species. Some young haplotypes were locally shared in mountainous areas that may have been shared refugia. The local exchange of cpDNA resulted in an excess of similar haplotypes between nearby populations. Our study demonstrated that the haplotype sharing pattern among East Asian Cerris oaks reflected the imprints of both shared ancestral polymorphism and introgression. This pattern was also associated with the relatively stable climates and complex landscapes in East Asia, which not only allowed the long‐term persistence of ancestral lineages but also connected the survived populations across refugia.


| INTRODUC TI ON
Extensive sharing of chloroplast DNA (cpDNA) haplotypes is commonly observed among closely related angiosperms, which may be caused by both introgression and shared ancestral polymorphism (Acosta & Premoli, 2010;Heuertz et al., 2006;Nevill et al., 2014;Palmé et al., 2004;Vitelli et al., 2017). On one hand, cpDNA is expected to be more frequently introgressed than nuclear DNA (Currat et al., 2008;Petit & Excoffier, 2009). This is because, in most angiosperms, maternally inherited cpDNA is only dispersed through seeds and thus has less potential for intraspecific gene flow (Petit et al., 2005); the low level of intraspecific gene flow hinders intraspecific homogenization. When two sister species come into contact, alien haplotypes from the invading species would not be diluted immediately by those of resident species (Currat et al., 2008;Du et al., 2011). Instead, they would be rapidly fixed in local populations under the enhanced effect of drift because haploid cpDNA has a much lower effective population size than diploid nuclear DNA (Currat et al., 2008;Herrera-Arroyo et al., 2013;Palmé et al., 2003).
These factors determine that chloroplast haplotypes are more likely to be shared among sister species through introgression (Acosta & Premoli, 2010). On the other hand, interspecific sharing of randomly distributed haplotypes may be a result of retained ancestral polymorphism (McGuire et al., 2007). According to the population genetics theory, population subdivision could increase a species' effective size, especially for cpDNA markers that experience low rates of migration (Hartl & Clark, 2007). This hints that ancestral lineages of cpDNA may be lost by drift less rapidly than nuclear DNA (Petit & Excoffier, 2009;Zhou et al., 2010). Furthermore, the low mutation rate of cpDNA may also decelerate lineage sorting and result in extensive sharing of ancestral haplotypes among sister species (Wolfe et al., 1987;Zhou et al., 2010). Given that cpDNA variation patterns more likely reflect the imprints of introgression (secondary contact) and shared geographic origin (ancestral sympatry) than speciation processes, it is essential to use information from as many closely related species as possible to track the evolutionary history of maternal lineages in angiosperms (Simeone et al., 2016).

Quercus (Fagaceae) is an ecologically important woody genus
with more than 400 species spread throughout the Northern Hemisphere . Plastid phylogeny of oaks is in general decoupled from taxonomy, which may be caused by both shared ancestral polymorphism and ancient hybridization among ancestral populations (Simeone et al., 2016;Yang et al., 2021). For example, Ilex oaks (sect. Ilex) were resolved as non-monophyletic using cpDNA data. Some Ilex oak species form the first diverging plastid lineage of the 'Old World oak' clade (subgenus Cerris), while the others are clustered with either Cerris oaks (sect. Cerris) or ring-cupped oaks (sect. Cyclobalanopsis). A fourth lineage has been found in the westernmost populations of the Mediterranean Q.
ilex (Simeone et al., 2016;Vitelli et al., 2017;Tekpinar et al., 2021;Yang et al., 2021;Zhou et al., 2022). A recent study has shown that shared ancestry alone is insufficient to explain the complex pattern; ancient hybridization also plays an important role . Interspecific sharing of chloroplast haplotypes is more frequently observed among sympatric oaks within the same section (e.g., Belahbib et al., 2001;Cavender-Bares et al., 2015;Dumolin-Lapègue et al., 1999;Lyu et al., 2018;Petit et al., 1997;Simeone et al., 2016;Pham et al., 2017;Whittemore & Schaal, 1991;Zhang, Hipp, & Gailing, 2015). This is particularly evident in European temperate white oaks (sect. Quercus), which share six lineages partitioned along a longitudinal gradient, reflecting a common history of long-term isolation among separate refugia and massive introgression during the post-glacial northward recolonization (Petit, Brewer, et al., 2002;Petit et al., 2004). However, the strong phylogeographic structure of European temperate white oaks is not found in eastern North American white oaks, suggesting a distinct biogeographic history in which past gene exchange occurred among populations from more diffuse refugia Pham et al., 2017).
East Asia is home to a quarter of the world's oak tree species, but patterns of haplotype sharing between native oaks are largely unknown (Lyu et al., 2018;San Jose-Maldia et al., 2017;Yang, Di, et al., 2016;Zeng et al., 2011). In this study, we explored the roles of shared ancestral polymorphism and introgression in shaping the haplotype sharing pattern among three closely related East Asian oaks, Quercus acutissima, Q. variabilis, and Q. chenii. Both nuclear and plastid data support that these species constitute a monophyletic group within sect. Cerris sister to the remaining members of the section in western Eurasia (Simeone et al., 2018;Hipp et al., 2020;Zhou et al., 2022). The divergence among the three species was estimated to have occurred during the early Oligocene to late Miocene . Currently, Q. acutissima and Q. variabilis are among the dominant elements of East Asian temperate deciduous forests, while Q. chenii is restricted to deciduous broad-leaved forests in eastern subtropical China (Huang et al., 1999). Morphological features including leaf and acorn size and distributions of leaf trichomes are used to distinguish them (Huang et al., 1999). Nevertheless, the occurrence of individuals with an intermediate phenotype suggests that putative hybrids may have occurred in their overlapping ranges (Liu, 1992;Hiroki & Kamiya, 2005).
Based on nuclear DNA data, recent studies have confirmed that the three closely related species are genetically coherent across their ranges despite introgressive hybridization Fu et al., 2022;Li et al., 2022;Liang et al., 2022). The admixture pattern between Q. acutissima and Q. chenii was affected by past climate shifts. Most putative hybrids were concentrated in an ancient contact zone that may have existed during the mid-Pliocene Warm Period but disappeared since the Early Pleistocene .
For Q. acutissima and Q. variabilis, strong signals of introgression were detected in sympatric populations throughout their ranges.
Ecologically similar populations tended to share more introgressed regions of the oak genome (Fu et al., 2022). Compared with nuclear DNA, cpDNA is less effective in discriminating the three closely related species (Zhang et al., 2020). The extensive sharing of ancestral haplotypes resulted in an extremely low level of cpDNA differentiation between Q. acutissima and Q. chenii . The wide distribution of these haplotypes also led to a non-significant phylogeographic structure for each of the three species (Chen et al., 2012;Li et al., 2019;Zhang, Li, et al., 2015). More interestingly, a recent study has shown that some narrowly distributed haplotypes were private to sympatric populations where admixed individuals between Q. acutissima and Q. chenii are frequently observed, suggesting that introgression also plays an important role in shaping the haplotype sharing pattern at a local scale . For these reasons, we infer that both shared ancestry and introgression may have left imprints on the phylogeographic patterns of East Asian Cerris oaks.
Here, we used two chloroplast intergenic spacers (atpB-rbcL and trnH-psbA) to examine the geographic patterns of chloroplast diversity in Q. acutissima and Q. chenii. The results were compared to cpDNA data previously obtained for Q. variabilis (Chen et al., 2012).
Our two specific objectives were as follows: (1) to explore the roles of shared ancestral polymorphism and introgression in shaping the haplotype sharing patterns among the three species; (2) to assess the influence of East Asian climates and landscapes on the phylogeographic history of East Asian Cerris oaks.  Table S1). Fresh and healthy leaves from 5 to 19 adult individuals spaced >50 m apart were collected at each sampling site and stored in silica gel. Total genomic DNA was extracted from 30 mg of dry leaf tissue of each individual using the Tiangen Plant Genomic DNA Kit (Tiangen, Beijing, China).
We compiled previously published sequence data of the same two regions for 41 populations of Q. variabilis (n = 439 individuals) sampled throughout its entire distribution in China (Figures 1 and S1, Table S1; Chen et al., 2012). We also obtained sequence data  (Simeone et al., 2016(Simeone et al., , 2018. However, sequence data from the same individual were not available for the four species in GenBank. For this reason, we reconstructed haplotypes for them using sequences belonging to the same species but different individuals. For the trnH-psbA region, we chose the most common haplotype of Q. cerris and Q. suber (Simeone et al., 2013(Simeone et al., , 2018 and the most common haplotype of the 'Cerris-Ilex' lineage of Q. coccifera and Q. ilex (Simeone et al., 2016). We selected the 'Cerris-Ilex' lineage for Mediterranean Ilex oaks because it is more closely related to Cerris oaks (Simeone et al., 2016). For the atpB-rbcL region, we used the only available sequences in GenBank, which were collected from planted trees of the four species in the Botanical Garden of Zurich, Switzerland (Kamiya et al., 2003). For the other outgroups, we extracted trnH-psbA and atpB-rbcL sequences from corresponding complete chloroplast genomes reported previously (Li et al., 2021;Liu et al., 2019;Pang et al., 2019;Yang et al., 2017;Yang, Zhou, et al., 2016, 2018. Finally, a total of 1053 concatenated sequences were analyzed in this study, including five species of sect. Cerris, nine species of sect. Ilex, and two species of sect. Cyclobalanopsis.

GenBank accession numbers of all the haplotypes detected in East
Asian Cerris oaks and all the 13 outgroups were provided in Table S2.

| Sequence variation and haplotype relationships
Sequences were proofread, aligned, and adjusted manually using BioEdit 7.2.5 (Hall, 1999). Insertions and deletions (indels) were treated as single mutation events and coded as substitutions (A/T) according to the simple gap coding method (Simmons & Ochoterena, 2000) as implemented in GapCoder (Young & Healy, 2003). An inversion of 32 bp detected in the trnH-psbA region was replaced with its reverse complement and coded as a substitution, comparable with an indel (Xu et al., 2015). Length variations in mononucleotide repeats were excluded because of their tendency for homoplasy (Qiu et al., 2009). The resulting alignments were concatenated into a single matrix using FasParser 2.1.1 (Sun, 2017).

| Genetic diversity, differentiation, and demographic history
The number of haplotypes (h), haplotype diversity (H d ), and nucleotide diversity (π) were calculated for each population using DnaSP 5.10. Average within-population gene diversity (h S ) and total gene diversity (h T ) were computed for each species using Permut 2.0 (Pons & Petit, 1996). The presence of phylogeographic structure was assessed by testing the difference between genetic differentiation among populations (G ST ) and the equivalent coefficient of differentiation considering similarities among haplotypes (N ST ). A higher N ST than G ST usually indicates the existence of a phylogeographic structure, that is, closely related haplotypes occur more often in the same populations than less related ones (Pons & Petit, 1996). The significance of the difference between G ST and N ST was tested by a permutation test (n = 10,000) in Permut 2.0. To further investigate interspecific differentiation, a hierarchical analysis of molecular variance (AMOVA) was performed with Arlequin 3.5 (Excoffier & Lischer, 2010). This analysis partitions the total genetic variance into three levels: among species, among populations within species, and within populations. The significance of variance components and their associated fixation indices (F CT , F SC , and F ST ) was assessed by 10,000 random permutations.

| Spatial autocorrelation
To investigate the spatial genetic structure at different geographic distance classes, we performed spatial autocorrelation analyses based on individual-level geographic and haplotype genetic distance matrices.
The first distance class was 0-50 km and the size of the following distance classes was increased in increments of 50 km. The significance of the autocorrelation coefficient (r) was tested for each distance class using a permutation test (n = 9999) that randomly shuffles all the individuals among sites. If the observed r-value lies beyond the upper 95% bound of the null distribution of permuted r values, a positive spatial genetic structure is inferred. The significance of r was also assessed by bootstrap resampling (n = 9999) with replacement from the original dataset for a specific distance class. When the 95% confidence intervals (CIs) do not overlap zero, a significant spatial genetic structure is inferred. These analyses were conducted for each species pair and all three species using GenAlEx 6.5 (Peakall & Smouse, 2012).

| Geographic pattern of interspecific cpDNA sharing
To test the hypothesis that nearby populations have a higher degree of interspecific cpDNA sharing than distant ones, we first calculated the gene identity (J), a measure of between-population genetic similarity, for all pairs of populations belonging to different oak species. We then compared the distributions of J among three groups of population pairs: (1) population pairs separated by <300 km and sharing haplotypes (J 1 ); (2) population pairs separated by <300 km, regardless of whether haplotypes were shared or not (J 2 ); and (3) population pairs separated by ≥300 km (J 3 ). We used 300 km as a threshold distance because we found that, for each species pair, the spatial autocorrelation coefficient declines smoothly when the size of the distance class exceeds 300 km, suggesting that the oak trees separated by <300 km were more genetically similar to each other.
The measures of interspecific gene identities and their means (M 1 , M 2 , and M 3 corresponding to J 1 , J 2 , and J 3 ) were computed according to Dumolin-Lapègue et al. (1999) and Belahbib et al. (2001). The distributions of J were compared statistically using a Wilcoxon ranksum test. All the analyses were performed for each species pair and all three species using R 3.5.1 (R Core Team, 2018).
The median-joining network grouped the 27 haplotypes of East Asian Cerris oaks into three non-species-specific lineages ( Figure 1d).
Among those, lineages A and B were consistently separated by a transition (A/G) in the atpB-rbcL region; lineages B and C differed by an 8-bp indel in the trnH-psbA region (Figure 1d; Table S3).
Haplotypes shared among the three species were often detected at a relatively high frequency and the internal parts of the network, such as H2 (4.0% of all the sampled individuals) and H7 (15.9%) in the lineage A, H1 (38.8%) in the lineage B, and H6 (10.8%) in the lineage C ( Figure 1d; Table S4). In contrast, haplotypes private to a single species were always found to be at a relatively low frequency and the tips of the network, such as H19 (1.4%) and H26 (0.3%) in the lineage A, H10 (0.4%) and H25 (0.1%) in the lineage B, and H12 (0.4%) and H22 (0.2%) in the lineage C (Figure 1d; Table S4). The lineage comprising all the 13 outgroups was separated by the lineage C by another 8-bp indel in the trnH-psbA region (Figure 1d; Table S3). Among the outgroups, two East Asian Ilex oaks (Q. dolicholepis and Q. baronii) and two reconstructed members of western Eurasian Cerris oaks (Q. cerris and Q. suber) shared the haplotype closest to the lineage C (i.e., H28). The other nine outgroups were separated from H28 by one to three mutational steps in the trnH-psbA (Q. bawanglingensis) or atpB-rbcL region (Q. phillyraeoides and Q. ilex), or by three to 10 mutational steps in both regions (the other six outgroups).
The three lineages of East Asian Cerris oaks presented an overlapping distribution (Figures 1 and S2-S4

| Genetic diversity, differentiation, and demographic history
The number of haplotypes (h), haplotype diversity (H d ), and nucleotide diversity (π) in each population ranged from 1 to 6 (mean 1.65), zero to 0.842 (mean 0.163), and zero to 0.00263 (mean 0.00032), respectively (Table S1). There were neither significant linear nor quadratic associations of genetic diversity with latitude and longitude (all p-values >.05). Among the three East Asian Cerris oaks, the total gene diversity (h T ) was found to be three to nine times greater than the average within-population gene diversity (h S ). The highest value of h T was observed in Q. chenii (0.902), followed by Q. variabilis (0.793) and Q. acutissima (0.747). The largest value of h S was detected in Q. acutissima (0.252), followed by Q. variabilis (0.120) and Q. chenii (0.100) ( Table 1).
For each species, genetic differentiation among populations was substantial as indicated by the high values of G ST and N ST (Table 1).
However, comparisons of these two measures did not reveal any sig-  Table 2). Most of the total genetic variation (68.79-82.45%) was partitioned among populations within species ( Table 2). The BSP showed that the three species maintained a relatively stable population size since the Middle Pleistocene ( Figure S32).

| Geographic pattern of interspecific cpDNA sharing
We compared interspecific gene identities among three groups of population pairs to test whether nearby populations have a higher level of interspecific cpDNA sharing than distant ones. When the most widespread haplotype H1 was considered, there was no significant difference between the mean of interspecific gene identities for population pairs separated by <300 km (M 2 ) and that for population pairs separated by ≥300 km (M 3 ) (P 23 ≥ 0.05 for all species pairs; Table 3). However, when H1 was excluded, M 2 was found to be 1.9-3.3 times greater than M 3 for the 'Q. acutissima-Q. variabilis' and 'Q.
Additionally, the mean of interspecific gene identities for population pairs separated by <300 km and sharing haplotypes (M 1 ) was found to be always significantly larger than M 2 and M 3 for all species pairs (P 12 and P 13 ≤ 0.004), regardless of whether H1 were considered or not ( Table 3).
Thirteen of the 39 population pairs shared five haplotypes with a narrow distribution (i.e., H8, H14, H15, H16, and H18; Figure 4; In these analyses, we also considered high-frequency haplotypes H2/H7 (lineage A) and H6 (lineage C) because the possibility of introgression-induced sharing of them cannot be ruled out. Twentysix of the 39 population pairs shared these three haplotypes (Figure 4;

| DISCUSS ION
We investigated the cpDNA variation in East Asian Cerris oaks based on the sequence data from 93 wild populations sampled throughout China. We found that the level of interspecific differentiation at cpDNA markers (F CT = 0.03; Table 2) is much lower than that reported for nuclear markers (F CT ≥0.15; Li et al., 2022;Liang et al., 2022). These results indicate that cpDNA is less efficient in discriminating the three closely related oaks. The extensive interspecific sharing of cpDNA haplotypes may arise from convergence, past and ongoing introgression/ hybridization, and retention of ancestral polymorphism (Acosta & Premoli, 2010;Nevill et al., 2014;Palmé et al., 2004). In our case, convergence seems to be unlikely because identical mutations Note: N, number of pairs of populations; M 1 , M 2 , and M 3 , means of interspecific gene identities for population pairs separated by <300 km and sharing haplotypes (J 1 ), for population pairs separated by <300 km (J 2 ), and for population pairs separated by ≥300 km (J 3 ); P ij , p-values for comparison between the distributions of J i and J j using Wilcoxon rank-sum test.

F I G U R E 4
Geographic distribution of nearby populations (separated by <300 km) belonging to different oak species and sharing chloroplast DNA (cpDNA) haplotypes except for H1. Red, blue, and yellow points represent populations of Quercus acutissima, Q. chenii, and Q. variabilis, respectively (see Table S1 for population codes). The colors of line segments represent the haplotypes shared by the populations at two endpoints.
are low-probability events and more than 40% of the haplotypes were shared by at least two species. Furthermore, all Fagaceae (and most Fagales) plastomes are notably conserved, that is, have generally low mutation fixation rates (Simeone et al., 2016;Yang, Hu, et al., 2018). The latter two explanations are possible because both randomly and locally distributed haplotypes were interspecifically shared, coinciding with the expectations of shared ancestry and introgression, respectively .

| Retention of ancestral polymorphism explains the sharing of randomly distributed haplotypes among East Asian Cerris oaks
Our analyses revealed three non-species-specific plastid lineages in East Asian Cerris oaks (Figure 1): the most ancestral lineage C is concentrated along the central Chinese mountain ranges; lineage B and its derivate, lineage A are widespread. A similar topology was also observed in the network generated for a single species or a pair of species using more cpDNA markers (see Table S6 for relationships among haplotypes identified in previous work ;Chen et al., 2012;Li et al., 2019Li et al., , 2022Zhang, Li, et al., 2015). In these studies, the divergence among lineages A, B, and C was supported by more parsimony-informative sites in the trnS (GCU) -trnG (UCC) and trnS (GCU) -trnT (GGU) regions. These non-species-specific lineages hint that the initial differentiation of plastid sequences of East Asian Cerris oaks is independent of the speciation process that formed the modern-day species; the three modern-day East Asian Cerris oaks share an ancestral plastid gene pool, which may have split into three lineages before the formation of modern species (Koch & Matschinger, 2007;Premoli et al., 2012;Simeone et al., 2016;Vitelli et al., 2017).
The internal haplotypes of the three major plastid lineages, H2/ H7 (lineage A), H1 (lineage B), and H6 (lineage C), presented a relatively high frequency and a wide geographic distribution across populations of the three species (Figure 1), coinciding with the expectation of shared ancestral polymorphism (McGuire et al., 2007).
According to the coalescent theory, common internal haplotypes that have more mutational connections and broader geographic distributions are more likely to be ancestral haplotypes (Posada & Crandall, 2001). If all the four haplotypes were present in the common ancestor and had been inherited by the extant taxa, they are expected to be randomly and widely distributed throughout the ranges of the descendant species (McGuire et al., 2007;Zhou et al., 2010;Zhang et al., 2013).
Compared with H1, the two internal haplotypes of the lineage A (H2/H7) are less frequent (Table S4). Previous studies have shown that in the network generated by more cpDNA markers, both H2 and H7 split into several separate haplotypes that were derived from a common missing ancestral haplotype (Table S6; Li et al., 2022). These results indicate that H2 and H7 themselves may only reflect part of the ancestral distribution of the lineage A. Within the H2 lineage, the ancestral type was only found in southwestern China, while the derived type was detected in both northwestern and southeastern China Zhang, Li, et al., 2015). Such a pattern suggests that the ancestor of the H2 lineage may have migrated from southwestern China to other regions. During this process, the H2 lineage may have diverged among multiple refugia , thus leaving a scattered distribution throughout China. In contrast, the H7 lineage was mainly detected in central and eastern China (Figure 1), of which one member was fixed in all the populations of Q. variabilis in Japan (Chen et al., 2012). Such a result implies that the ancestor of the H7 lineage may have diverged locally in central and eastern China and further expanded eastward to Japan probably through the East China Sea land bridge during the glacial periods (Sakaguchi et al., 2012;Wang et al., 2022).
Among the three lineages, the lineage C was most directly Mediterranean oaks (e.g., Simeone et al., 2018;Vitelli et al., 2017) can be ruled out: all haplotypes, including the putatively most ancestral lineage C are restricted to East Asian members of sect. Cerris.
Consistent with our results, Zhang et al. (2020) showed that complete chloroplast genome sequences of three Q. acutissima trees did not cluster together. Indeed, one of those from northeastern China presented the haplotype H7 (Zhang et al., 2020), while the other two from northwestern and eastern China shared H1 with two Q. variabilis and Q. chenii individuals Yang, Hu, et al., 2018). Thus, it is reasonable to see that some Q. acutissima trees were grouped with heterospecific trees, rather than conspecific trees, supporting our conclusion that the plastid phylogeny of East Asian Cerris oaks is decoupled from taxonomic boundaries.
The locally shared haplotypes were largely concentrated in mountainous areas of central and eastern China (Figure 4). Previous studies have shown that these mountainous areas may have been glacial refugia for many temperate and subtropical tree species (Qiu et al., 2011), including Q. acutissima Zhang et al., 2018), Q. variabilis (Chen et al., 2012), and Q. chenii . These results suggest that the three oak species may have had multiple isolated but shared glacial refugia, thus allowing long-term coexistence and historical introgression in different areas Soliani et al., 2012;Thomson et al., 2015). Indeed, the persistence of East Asian Cerris oaks in these regions may be traced back to an earlier time as evidenced by the rich and widespread fossil records dating to the Miocene and Pliocene (e.g., Shanwang in northern China and Mingguang in southeastern China; Barrón et al., 2017;Momohara, 2016;Song et al., 2000;Zhou, 1993). Such a long history would provide sufficient time for East Asian Cerris oaks to share plastid genomes through introgression. Using nuclear markers, a recent study has shown that a mid-Pliocene contact zone between the already isolated Q. acutissima and Q. chenii may have occurred in central and eastern China, where more than 70% of the putative hybrids were concentrated . Furthermore, a relatively high level of introgression was also detected in sympatric populations of Q. acutissima and Q. variabilis in central and eastern China (Fu et al., 2022). These results support our inference that interspecific sharing of narrowly distributed haplotypes in this region is more likely to be a result of introgression. individual of the Q. acutissima population at the Tianmu Mountain, a forest rich in Tertiary relicts (Wang, 1961). The same haplotype is private to the northeasternmost Q. variabilis population in our data (Liaodong Peninsula), where it's again shared by one out of 12 individuals of nearby Q. acutissima populations. That the same satellite haplotype evolved in a northeastern Q. variabilis and two ~1100 km afar southeastern Q. chenii populations may be due to fixation of a convergent mutation. However, in the overall conservation of plastid DNA in oaks, it may as well represent a common geographic origin.
Once more widespread, the now-extinct northeastern Q. chenii population was introgressed by Q. variabilis in northeastern China and its private plastome was captured. Q. acutissima captured this potential Q. chenii plastome when introgressing at a large scale into its sibling species.
The local sharing of haplotypes may also be influenced by demographic history and the complex landscapes of East Asia. During the Pleistocene, local plants like oaks underwent repeated range contractions and expansions (e.g., Fan et al., 2018;Tian et al., 2015;Ye et al., 2018). These events would increase the level of genetic admixture and promote the spread of some locally distributed haplotypes across multiple refugia . Additionally, the geographic ranges of the three species encompass a complex landscape made up of numerous north-south and east-west oriented mountain ranges (Tang et al., 2006). These mountains have not only provided multiple marginal refugia but also offered dispersal corridors for range expansions, thus allowing the migration of individuals with the same haplotypes between different refugia in response to Pleistocene climatic fluctuations (Tian et al., 2018).
Our findings support that local exchange of chloroplast genomes results in an excess of similar haplotypes between nearby populations from different species (Table 3). However, such an effect was not found in the 'Q. chenii-Q. variabilis' pair, indicating that the level of local introgression differs among species pairs, probably related to the degree of co-occurrence in the overlapping ranges (Dumolin-Lapègue et al., 1999), today or in the past. Indeed, we observed only a few cases where Q. chenii and Q. variabilis coexist in the wild (e.g., the Lushan Mountains, Jiangxi Province) and our dataset did not include any pair of populations that belong to these two species and are separated by less than 1 km. In contrast, mixed stands of Q. acutissima and Q. chenii or Q. variabilis are commonly observed in the field. The interspecific gene identities for sympatric populations of these two species pairs (0.62; Table S7)

| CON CLUS IONS
Our study demonstrates that the haplotype sharing pattern among East Asian Cerris oaks reflects the imprints of both shared ancestral polymorphism and repeated phases of secondary gene flow/reticulation via introgression/hybridization. The three major plastid lineages presented an overlapping distribution, especially in central and eastern China, which differs from that of other Fagales species in Europe and South America by lacking an obvious geographic westeast or north-south structuring. In these species, cpDNA lineages shared among closely related species are partitioned longitudinally or latitudinally, mirroring a history of introgression among multiple isolated refugia (Acosta & Premoli, 2010;Premoli et al., 2012). In contrast, East Asia is characterized by complex landscapes and relatively stable climates, which not only allowed the long-term persistence of ancestral lineages but also connected the survived populations across refugia (Qiu et al., 2011;Tang et al., 2006;Tian et al., 2018). These factors contribute to the overlapping distribution of shared plastid lineages among East Asian Cerris oaks.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
Sequence data of East Asian Cerris oaks are available on GenBank Yanming Fang https://orcid.org/0000-0003-2320-9539